Project Summary The signaling lipid sphingosine 1-phosphate (S1P) plays critical roles in the immune system. Notably, S1P regulates lymphocyte exit from lymph nodes, where lymphocytes are initially activated to fight pathogens, out into lymph, which carries the cells to blood and enables them to travel to infected tissues. FTY720, a drug that targets four S1P receptors including S1PR1, became the first FDA-approved oral therapeutic for multiple sclerosis. By blocking pathogenic T cells from exiting lymphoid organs, FTY720 prevents them from accessing the central nervous system. Second-generation drugs that target S1PR1 have also shown promise in Phase II clinical trials for psoriasis and colitis. Unfortunately, these drugs also target S1PR1 in endothelial cells and cardiomyocytes, resulting in serious side effects including macular edema and bradycardia. We found that the major facilitator superfamily transporter SPNS2 supplies S1P into lymph but not blood, making it a promising new drug target that would enable spatially specific modulation of S1P signaling. Furthermore, our data indicate that Spns2 is required for the accumulation of activated T cells at sites of inflammation and deleting Spns2 has a protective effect in experimental autoimmune encephalomyelits, a mouse model of multiple sclerosis. Based on these observations, we set out to develop a high throughput screen to identify small molecule modulators of Spns2 activity. We developed and optimized a Rapid-Fire high-throughput mass spectrometry-based assay to detect S1P secreted into the media by Spns2-expressing cells. Utilizing this assay, we screened Evotec's 252,454 compound Discovery Library, yielding 5,899 inhibitors at a cut-off of 11.5% (5?). We subsequently cherry picked 1962 hits for confirmation screens, yielding 923 confirmed hits. Here, we propose to develop assays and validate the hits that emerged from the HTS effort. Together with NYU's Office of Therapeutics Alliances we have identified a team of consultants and contract research organizations with expertise in assay development and screening, hit validation, and synthetic and medicinal chemistry that will work with our laboratory to develop and execute a hit validation scheme yielding high quality probes targeting Spns2. These tool compounds can be used to probe the biology of Spns2 and have the potential to yield first in class drugs for treating autoimmune diseases.